Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

IntroductionCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored.MethodsImmune cells of the small intestinal Peyer’s patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals.ResultsHere, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection.DiscussionConsidering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host–Cryptosporidium interactions in target species

Establishment of a Newborn Lamb Gut-Loop Model to Evaluate New Methods of Enteric Disease Control and Reduce Experimental Animal Use

Enteric infectious diseases are not all well controlled, which leads to animal suffering and sometimes death in the most severe cases, in addition to economic losses for farmers. Typical symptoms of enteric infections include watery diarrhea, stomach cramps or pain, dehydration, nausea, vomiting, fever and weight loss. Evaluation of new control methods against enteric infections requires the use of many animals. We aimed to develop a new method for an initial in vivo screen of promising compounds against neonatal diseases such as cryptosporidiosis while limiting experimental animal use. We therefore adapted an in vivo method of multiple consecutive but independent intestinal loops to newborn lambs delivered by cesarean section, in which endotoxin responsiveness is retained. This new method allowed for the screening of natural yeast fractions for their ability to stimulate immune responses and to limit early Cryptosporidium parvum development. This model may also be used to investigate host–pathogen interactions and immune responses in a neonatal controlled environment

Immunostimulation of newborn animals with yeast-derived products: application to cryptosporidiosis control

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Impact d’une infection intestinale pendant la période néonatale par Cryptosporidium parvum sur la sensibilité à une infection par Heligmosomoides polygyrus bakeri ou Salmonella enterica à l’âge adulte

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Characterization of intestinal macrophages and dendritic cell subsets in neonatal lambs at homeostasis and following Cryptosporidium parvum infection

International audienceCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in livestock (cattle, sheep, goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and in particular of CD11c+CD103+CD11b-conventional dendritic cells (cDC) among mononuclear phagocytes (MP) in controlling the acute phase of Cp infection. During infection, in response to chemokine production by infected epithelial cells, newly recruited cDCs produce IL12 and IFNγ contributing to the elimination of the parasite. According to the well-established mouse cDC classification, this Batf3+DC subpopulation corresponds to the cDC1 subset. The aim of this project was to better characterize intestinal MP subpopulations in neonatal lamb and calf at homeostasis and during Cp infection. As in the mouse model, the parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. MP were therefore analyzed in lymphoid and non-lymphoid intestinal tissues of lambs and claves. We performed phenotypic and functional analyses of mononuclear phagocytes by flow cytometry and by transcriptomic methods (FLUIDIGM®) respectively, in the distal jejunum, jejunal and ileal Peyer’s patches. We characterized a population of macrophages and three subpopulations of cDC. We demonstrated that the subset identified as cDC1, according to the current common classification of cDC in different species (human, mouse, pig, sheep and chicken), increases with the age of animal. This might be linked with the decrease in sensitivity to C. parvum observed with the age. We are currently investigating the evolution of cDC1 subset during C. parvum infection

Impact of early-life exposure to <em>Cryptosporidium parvum</em> infection on intestinal homeostasis at adulthood

National audienceNumerous studies recently describe the relationship between influences during early-life period and later-life health and disease. Cryptosporidium parvum is a zoonotic parasite responsible for a diarrheal disease that affects mostly children under 5 and immunocompromised patients (AIDS or organ transplant patients). Epidemiological studies have reported that after resolution of C. parvum infection, patients still suffer for abdominal pain. In this context, by using a neonatal mouse model of cryptosporidiosis, we tried to decipher the intestinal consequences at adulthood of the neonatal infection by analyzing the composition of the microbiota, the composition of immune cells in the intestine but also consequences on visceral sensitivity and the susceptibility to an unrelated intestinal infection. We observed that adult mice infected by C. parvum during the neonatal period display a modification of microbiota and of the composition of immune cells. These intestinal modifications were associated with an increased viscero-sensitivity and with a higher sensitivity to Salmonella infection. Altogether these results clearly demonstrate that an infection by C. parvum during the neonatal period induces intestinal imprinting that can be responsible for abdominal pains and increased susceptibility to another intestinal infection, way after the resolution of C. parvum infection

Characterization of Intestinal mononuclear phagocyte subsets of young lamb at homeostasis by single cell RNA-Seq and during Cryptosporidium parvum infection by flow cytometry.

International audienceIntestinal mononuclear phagocytes are key immune cells that maintain intestinal homeostasis and participate in the protective immune responses toward pathogens. Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in ruminant farms. Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity in controlling the acute phase of Cp infection and deciphered the role of different subsets of intestinal mononuclear phagocytes in this protective immune response. The aim of this project was to better characterize intestinal mononuclear phagocytes in lamb at homeostasis and during Cp infection. The parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (IPP) (lymphoid tissue) that extends all along the ileum. We first performed a characterization of mononuclear phagocytes present in the IPP of a non-infected 10-day-old lamb by a single cell transcriptomic approach on CD11c+ MHCII+ sorted cells. This global approach allowed us to identify in the IPP of young lamb two main populations of macrophages and at least three different population of dendritic cells. Then, we carried out phenotypic and functional analyses of these different cell subsets by flow cytometry and transcriptomic methods in various compartments of the small intestine at homeostasis and during infection

Intestinal mononuclear phagocyte subsets of young ruminants : Characterization at homeostasis and during Cryptosporidium parvum infection

International audienceCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in ruminant farms. Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we demonstrated the importance of the innate immunity and in particular of CD11c+CD103+CD11b- dendritic cells (DC) in controlling the acute phase of Cp infection. During infection, in response to chemokine production by infected epithelial cells, newly recruited CD11c+CD103+CD11b- DC produce IL12 and IFNγ contributing to the parasite elimination process. According to the common classification of DC in different species (human, mouse, pig, sheep), this subpopulation can be identified as the cDC1 subset of conventional DC. The aim of this project is to better characterize intestinal DC subpopulations in lamb and calf at homeostasis and during Cp infection. The parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. We therefore performed phenotypic and functional analyses of mononuclear phagocytes by flow cytometry and transcriptomic methods in various compartments of the small intestine. We characterized one population of macrophages and three subpopulations of DC. We demonstrated that the subset identified as cDC1 increases according to the age of animal, and this might be linked with the decrease in sensitivity to C. parvum observed with the age. We are currently investigating cDC1 subset during C. parvum infection

Characterization of the intestinal mononuclear phagocytic cells in the intestine of the lamb to better understand the immune response against Cryptosporidium parvum

International audienceCryptosporidiosis is a poorly controlled zoonosis caused by the intestinal infection by a protozoan parasite, Cryptosporidium parvum (Cp), presenting a high prevalence in ruminant farms (cattle, sheep, goats). Young animals with an immature intestinal immune system are particularly susceptible to this infection. In a neonatal mouse model, we previously demonstrated the key role of innate immunity and in particular of CD11c+CD103+CD11b- dendritic cell (DC) subset incontrolling the acute phase of Cp infection [1]. During infection, in response to chemokine production by infected epithelial cells, newly recruited CD11c+CD103+CD11b- DC produce IL12 and IFNγ contributing to the elimination process of the parasite. According to the current common classification of DC in different species (human, mouse, pig, sheep, and chicken), this subpopulation can be identified as the cDC1 subset of conventional DC. The aim of this project was to better characterize intestinal DC subpopulations in lamb and to determine their role during Cp infection. As in the mouse model, the parasite invades and multiplies mainly in the ileum of young lambs. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. We characterized in various compartments of the lamb small intestine (lymphoid and non-lymphoid) the various mononuclear phagocyte populations by performing a phenotypic and functional analysis by flow cytometry and transcriptomic methods respectively. We followed the evolution of the cell subsets according to the age of the animals and during Cp infection. This work represents the first fine description of mononuclear phagocyte cell subsets in the small intestine of young lambs at homeostasis and during the course of C. parvum infection

Importance of the intestinal cDC1 dendritic cells in the control of cryptosporidiosis in the murine and lamb models

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